The formation of DNA adducts, using the 32P-postlabelling assay, and induction of 7-ethoxyresorufin O-deethylase (EROD) were investigated in a primary culture of rainbow trout hepatocytes exposed to benzo[a]pyrene (B[a]P). Concentrations of 0.1 and 1 μm-B[a]P were shown to induce EROD whereas 10 μm was an inhibitory concentration. DNA adducts were detected for 12 hr to 72 hr after exposure to 1 μm-B[a]P whereas EROD activity was increased 36 hr after treatment. The pattern of adducts was shown to be identical to that obtained after B[a]P treatment of rainbow trout in vivo, as demonstrated by co-chromatography of the adducts. Pre-exposure of hepatocytes for 48 hr to β-naphthoflavone (βNF) and subsequent 24-hr exposure to 1 μm-B[a]P did not lead to increased DNA adduct formation although βNF treatment led to a 3.4-fold induction of EROD activity at the time of B[a]P addition. This study suggests that primary culture of rainbow trout hepatocytes provides a suitable method for studying DNA adduct formation and its modulating factors in vitro.